Avian leukosis virus subgroup J (ALV-J) infection can cause tumors and immunosuppression in infected chickens. Macrophages play a central role in host defense against invading pathogens. In this study, we discovered an interesting phenomenon: ALV-J replication is weakened from 3 hours post-infection (hpi) to 36 hpi, which was verified using Western blotting and RT-PCR. To further investigate the interaction between ALV-J and macrophages, transcriptome analysis was performed to analyze the host genes’ function in chicken primary monocyte-derived macrophages (MDM). Compared to the uninfected control, 624 up-regulated differentially expressed genes (DEG) and 341 down-regulated DEG at 3 hpi, and 174 up-regulated DEG and 87 down-regulated DEG at 36 hpi were identified in chicken MDM, respectively. ALV-J infection induced strong innate immune responses in chicken MDM at 3 hpi, instead of 36 hpi, according to the analysis results of Gene Ontology and KEGG pathway. Importantly, the host factors, such as up-regulated MIP-3α, IL-1β, iNOS, K60, IRG1, CH25H, NFKBIZ, lysozyme and OASL were involved in the host defense response during the course of ALV-J infection. On the contrary, up-regulated EX-FABP, IL4I1, COX-2, NFKBIA, TNFAIP3 and the Jak STAT pathway inhibitors including CISH, SOCS1 and SOCS3 are beneficial to ALV-J survival in chicken macrophages. We speculated that ALV-J tropism for macrophages helps to establish a latent infection in chicken MDM from 6 to 36 hpi. The present study provides a comprehensive view of the interactions between macrophages and ALV-J. It suggests the mechanisms of defense of chicken macrophages against ALV-J invasion and how ALV-J escape the host innate immune responses.

Our previous studies showed that microRNA-15a (miR-15a) was closely related to intramuscular fat (IMF) deposition in chickens; however, its regulatory mechanism remains unclear. Here, we evaluated the expression characteristics of miR-15a and its relationship with the expression of acetyl-CoA acyltransferase 1 (ACAA1), acyl-CoA oxidase 1 (ACOX1) and sterol carrier protein 2 (SCP2) by qPCR analysis in Gushi chicken breast muscle at 6, 14, 22, and 30 weeks old, where we performed transfection tests of miR-15a mimics in intramuscular preadipocytes and verified the target gene of miR-15a in chicken fibroblasts (DF1). The miR-15a expression level at 30 weeks increased 13.5, 4.5, and 2.7-fold compared with the expression levels at 6, 14, and 22 weeks, respectively. After 6 days of induction, miR-15a over-expression significantly promoted intramuscular adipogenic differentiation and increased cholesterol and triglyceride accumulation in adipocytes. Meanwhile, 48 h after transfection with miR-15a mimics, the expression levels of ACAA1, ACOX1 and SCP2 genes decreased by 56.52%, 31.18% and 37.14% at the mRNA level in intramuscular preadipocytes. In addition, the co-transfection of miR-15a mimics and ACAA1, ACOX1 and SCP2 3′UTR (untranslated region) dual-luciferase vector significantly inhibited dual-luciferase activity in DF1 cells. Taken together, our data demonstrate that miR-15a can reduce fatty acid oxidation by targeting ACAA1, ACOX1, and SCP2, which subsequently indirectly promotes the differentiation of chicken intramuscular preadipocytes.

Circular RNAs (circRNAs) play a significant regulatory role during skeletal muscle development. To identify circRNAs during postnatal skeletal muscle development in chickens, we constructed 12 cDNA libraries from breast muscle tissues of Chinese Gushi chickens at 6, 14, 22, and 30 weeks and performed RNA sequencing. In total, 2112 circRNAs were identified, and among them 79.92% were derived from exons. CircRNAs are distributed on all chromosomes of chickens, especially chromosomes 1–9 and Z. Bioinformatics analysis showed that each circRNA had an average of 38 miRNA binding sites, 61.32% of which have internal ribosomal entry site (IRES) elements. Furthermore, in total 543 differentially expressed circRNAs (DE-circRNAs) were identified. Functional enrichment analysis revealed that DE-circRNAs source genes are engaged in biological processes and muscle development-related pathways; for example, cell differentiation, sarcomere, and myofibril formation, mTOR signaling pathway, and TGF-β signaling pathway, etc. We also established a competitive endogenous RNA (ceRNA) regulatory network associated with skeletal muscle development. The results in this report indicate that circRNAs can mediate the development of chicken skeletal muscle by means of a complex ceRNA network among circRNAs, miRNAs, genes, and pathways. The findings of this study might help increase the number of known circRNAs in skeletal muscle tissue and offer a worthwhile resource to further investigate the function of circRNAs in chicken skeletal muscle development.

Background: Coloration is one of the most recognizable characteristics in chickens, and clarifying the coloration mechanisms will help us understand feather color formation. “Yufen I” is an commercial egg-laying chicken breed in China, that was developed by a three-line cross using lines H, N and D. Columbian plumage is a typical feather character of the “Yufen I” H line. To elucidate the molecular mechanism underlying pigmentation of Columbian plumage, this study utilizes the technology of high-throughput sequencing to compare the transcriptome and proteome differences in different feather follicular tissue, including the dorsal neck with black and white striped feather follicles (Group A) and the ventral neck with white feather follicles (Group B) in the “Yufen I” H line. Results: In this study, we identified a total of 21,306 genes and 5203 proteins in chicken feather follicles. Among these, 209 genes and 382 proteins were differentially expressed in two different locations, Group A and Group B, respectively. A total of 8 differentially expressed genes (DEGs) and 9 differentially expressed proteins (DEPs) were found to be involved in the melanogenesis pathway. Besides, a specifically expressed MED23 gene and a differential expressed GNAQ protein were involved in melanin synthesis. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis mapped 190 DEGs and 322 DEPs, to 175 and 242 pathways, respectively, and there were 166 pathways correlated with both DEGs and DEPs. 49 DEPs/DEGs overlapped and were enriched for 12 pathways. Transcriptomic and proteomic analyses revealed that the following pathways were activated: melanogenesis, cardiomycete adrenergic, calcium and the cGMP-PKG. The expression of DEGs was validated by real-time quantitative polymerase chain reaction (qRT-PCR) that was similar to that of RNA-seq. In addition, we found that MED23, FZD10, WNT7B and WNT11 genes expression peaked at approximately 8 weeks in the “Yufen I” H line, which is consistent with the molting cycle. As both the groups showed significant differences in terms of expression of the genes studied, this study opens up avenues for study in the future to assess their exact function in color of plumage. Conclusion: These common DEGs and DEPs were enriched in the melanogenesis pathway. The MED23 and GNAQ were also reported to have a crucial part synthesis of melanin. In addition, this study is the first to reveal variations in gene and protein in the “Yufen I” H line during Columbian feather color development, and discover principal genes and proteins that would aid in the functional genomics studies in future. The results of the present study provide a significant conceptual basis for the “Yufen I” H line future breeding schemes and provide a basis for research on the mechanisms of feather pigmentation.